cytokine-induced killer cells as an adoptive cellular...

Biochemistry and Molecular Biology 2019; 4(1): 6-16

http://www.sciencepublishinggroup.com/j/bmb

doi: 10.11648/j.bmb.20190401.12

ISSN: 2575-5064 (Print); ISSN: 2575-5048 (Online)

Cytokine-Induced Killer Cells as an Adoptive Cellular Immunotherapy Strategy for Hepatocellular Carcinoma

Nahla El-Sayed El-Ashmawy1, Enas Arafa El-Zamarany

2, Hoda Abd El-Kader El-Bahrawy

1,

Enas Abd El-Moneim Zahran1, *

1Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt 2Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Email address:

*Corresponding author

To cite this article: Nahla El-Sayed El-Ashmawy, Enas Arafa El-Zamarany, Hoda Abd El-Kader El-Bahrawy, Enas Abd El-Moneim Zahran. Cytokine-Induced

Killer Cells as an Adoptive Cellular Immunotherapy Strategy for Hepatocellular Carcinoma. Biochemistry and Molecular Biology.

Vol. 4, No. 1, 2019, pp. 6-16. doi: 10.11648/j.bmb.20190401.12

Received: March 13, 2019; Accepted: April 13, 2019; Published: May 17, 2019

Abstract: Background: Hepatocellular carcinoma (HCC) is the most common histologic type of primary liver cancer. HCC

is the second highest mortality rate out of all major malignant carcinomas worldwide. Objectives: The aims of this study were

to establish a rapid and easily handled culture method for sufficient expansion of viable and cytotoxic cytokine-induced killer

(CIK) cells against HCC. Also, this study aimed to examine the morphologic, phenotypic, and functional characteristics of CIK

cells. Method: Peripheral blood mononuclear cells (PBMCs) were cultured in a preliminary static culture to remove adherent

cells. The suspended cells were cultured for 14 days with interferon-γ, human monoclonal anti-CD3 antibody and

interleukin-2. Aliquots of induced PBMCs were harvested weekly to assess informative morphologic and phenotypic features

of CIK cells. Mature CIK cells were subjected to functional assays that included the production of TNFα and the cytotoxic

effect on HCC cell line, HepG2. Findings: CIK cells could be successfully expanded from all samples with a significant

increase in T cells, natural killer cells, and natural killer T cells. TNFα concentration in the culture supernatant was

significantly increased. The cytotoxic effect of CIK cells on HepG2 cells was nearly 60% at 40:1, effector: target ratio.

Regression analysis was used to predict the CIK: HepG2 ratio required to achieve complete cytotoxicity. Conclusion: This

study provides a detailed and simple strategy for culturing effective CIK cells. Mature CIK cells showed a high functional

capacity against HCC; which will support the further ongoing practice of immunotherapy integration into different current

cancer treatment protocols.

Keywords: Peripheral Blood Mononuclear Cells, CIK Cells, Natural Killer T Cells, Cytotoxicity, Liver Cancer

1. Introduction

Liver cancer is one of the very most commonly diagnosed

malignant tumors. It is the second highest mortality rate

worldwide; estimated to be responsible for 746,000 passings

in 2012 [1]. The most common histologic type of primary

liver cancer, hepatocellular carcinoma (HCC), is a malignant

tumor emerging from hepatocytes. Globally, in the vicinity of

600,000 and one million new cases of HCC are diagnosed

every year, with a survival rate ranged from 6 to 20 months

[2]. HCC is an assertive malignancy with a poor prognosis, in

which repeated hepatocyte damage sets up an endless loop of

cell apoptosis and regeneration that in the long run outcomes

in genomic instability and initiation of HCC [3].

HCC is affiliated with cirrhosis in 80–90% of cases.

Hepatitis B virus infection is the main risk factor for HCC,

globally, accounts for no less than half cases of HCC.

Hepatitis C virus infection is the second most common risk

factor, with a predicted 10–25% of all cases of HCC ascribed

to it worldwide. Other significant risk factors include

alcohol-induced cirrhosis, obesity, aflatoxin, fatty liver, iron

overload, diabetes, and smoking [4]. Recently, non-alcoholic

Biochemistry and Molecular Biology 2019; 4(1): 6-16 7

fatty liver and non-alcoholic steatohepatitis, emerging from

metabolic disorders such as insulin resistance syndrome, are

included [5]. The liver is a tolerogenic organ with unique

mechanisms of immune regulation. It has to anticipate

peculiar immune responses to gut-derived antigens that

steadily circulate through it [6].

Diagnosed cancers must have bypassed the body’s

antitumor immune responses to grow chronologically. Aside

from liver tolerogenic nature, the loss of tumor-associated

antigens, decreased major histocompatibility complex

(MHC) antigen expression, inactivation of T cells by reduced

T cell receptor signaling or IL-10, and transforming growth

factor-β-mediated suppression, cause a scene of immune

tolerance to tumors [7]. As the liver disease advances from

cirrhosis to HCC, many immune cells’ functions become

impaired. T cells, both helper and cytotoxic types, diminish

in numbers with constricted function and increased

expression of inhibitory receptors. T helper 17 cells increase

in number leading to increase tumor angiogenesis [8].

Cancer-associated fibroblasts inhibit natural killer (NK) cells

function. Myeloid-derived suppressor cells suppress T cell

stimulation, activate other immune-suppressive cells, and

stimulate tumor angiogenesis [9].

Traditional treatments available for HCC patient are

limited due to the advanced stage at which most patients are

diagnosed. Surgical resection is a decent decision for most

early-stage patients [10]. Sorafenib is a directed therapy,

which is the standard first-line systemic drug for advanced

HCC. However, patients with severe hepatic dysfunction or

poor performance status do not derive any survival perk from

this therapy [11]. Liver transplant is another perioperative

intercession for advanced cases of HCC; nonetheless, there

are limitations due to the deficient number of the matched

donors and post-transplant allograft rejection [10]. HCC is

extremely chemo-resistant as multi-drug resistance genes are

reported to be highly expressed in HCC [12].

Unlike, the non-selectivity of traditional treatments,

immunotherapy, hypothetically, could selectively target and

devastate malignant cells with insignificant side effects [12].

It appears to work better in more immunogenic tumors like

HCC. It is based on the redirection the patient’s own immune

system against cancer instead of targeting cancer itself (e.g.

by chemotherapy). Adoptive cell therapy (ACT) is one of the

major therapeutic options in cancer immunotherapy. It

involves the transfer of an expansive number of ex vivo-

cultured and functional immune cells into a tumor-bearing

host [13]. It has employed numerous types of immune cells,

including dendritic cells, cytotoxic T lymphocytes,

lymphokine-activated killers, NK cells, and cytokine-induced

killer (CIK) cells [14]. The ACT is a ‘living’ treatment in

light of the fact that the managed cells can proliferate in vivo

and keep up their antitumor effector functions. Such

immunological stimulation may offset the strongly immune-

suppressive microenvironment in the liver [15].

CIK cells, as the most frequently used ACT, are a

heterogeneous cell population including natural killer T

(NKT) cells (CD3+CD56

+), T cells (CD3

+CD56

−), and NK

cells (CD3−CD56

+) cells. Normally, the most cytotoxic cells

with double T/NK phenotype are uncommon but present (1%

to 5%) in circulating blood [16]; yet, after in vitro expansion

for 14 days, they may reach 20 to 30% of the total CIK cells

[17]. Most of these NKT cells have been shown to be derived

from the T cells and not from NK cells [18]. CIK cells have

advantages of a higher proliferation rate, MHC-unrestricted

activity,

a strong activity against tumors with minimal

toxicity and graft versus host disease [19], and exceptionally

important, they are not hindered by immune-suppressive

drugs [20]. Interferon-gamma (IFNγ) and tumor necrosis

factor-alpha (TNFα) are the principle cytokines produced by

CIKs [21].

Due to the number of advantages of CIK cells, they

present a promising immunotherapy approach that could be

used for HCC. Along these lines, the present study evaluates

the potential of in vitro expansion of viable CIK cells from

human peripheral blood mononuclear cells (PBMCs) and

measures the proportion of the most effective subset

CD3+CD56

+ in the culture. In addition, the study examines

TNFα secretion and the cytotoxicity of expanded CIK cells in

vitro on HepG2 cell line.

2. Materials and Methods

2.1. Reagents

Roswell Park memorial institute medium (RPMI)-1640

with L-glutamine, RPMI-1640 without L-glutamine or

phenol red, calcium-magnesium-free phosphate-buffered

saline (PBS), penicillin-streptomycin-amphotericin B

100IU/100µg/0.25µg/mL, and Trypan blue 0.4% exclusion

dye were all obtained from Lonza Verviers SPRL®, Belgium.

Ficoll® paque plus was obtained from Sigma-Aldrich

®,

United Kingdom. Fetal bovine serum (FBS) was obtained

from Biowest®, France. IFNγ and interleukin-2 (IL-2) were

obtained from PeproTech®, United Kingdom. Low endotoxin,

azide-free (LEAF™) purified anti-human CD3 antibody was

obtained from Biolegend®, USA. Monoclonal FITC-

conjugated anti-CD3 antibody, monoclonal PE-conjugated

anti-CD56 antibody, and fluorescence-activated cell sorting

buffer were obtained from BD Biosciences, USA. Cell

counting kit-8 (CCK-8) and TNFα picoline ELISA kit were

obtained from Boster Biological Technology®, USA. Heparin

sodium was obtained from the Nile Co®, Egypt.

2.2. Generation of CIK Cells

2.2.1. Blood Collection

Peripheral blood (PB) samples from 10 healthy volunteers

were collected by phlebotomy and emptied in sterile

centrifuge tubes (50 mL) containing 5000 I.U. heparin

sodium as anticoagulant followed by inversion several times,

to ensure proper mixing, and labeling of the samples.

2.2.2. PBMCs Separation

PB samples were diluted 1:1 with PBS and mixed well, as

dilution gives a better yield of mononuclear cells (MNCs)

8 Nahla El-Sayed El-Ashmawy et al.: Cytokine-Induced Killer Cells as an Adoptive Cellular Immunotherapy

Strategy for Hepatocellular Carcinoma

[22]. MNCs were separated by density gradient using cooling

swing out centrifuge (with no break) (Centurion®, United

Kingdom) for 30 minutes and 1400 rotation/minute (rpm).

Optimal centrifugation temperature (18-20°C) is required to

keep density gradient medium at 1.077 g/mL [23]. The

MNCs at the Blood-Ficoll®

interface were aspirated and

washed twice with 50 mL PBS, and then, washed once with

RPMI-1640 medium and resuspended in 1 mL medium. Total

MNCs (lymphocytes, monocytes, macrophages, and stem

cells) were counted using hemocytometer by means of trypan

blue exclusion dye.

2.2.3. Preliminary Static Culture of CIK Cells

Preliminary MNCs culture is required to remove adherent

cells and obtain the suspension lymphocyte portion of MNCs

(Figure 1); which would later be differentiated into CIK

cells. PBMCs were seeded at a density of 2×106 cells/mL and

were allowed to adhere to tissue culture flask (Corning®,

USA) containing 10 mL of RPMI-1640 medium for 2 hours

in a humidified incubator (Shel Lab®, USA) at 37

oC and 5%

CO2. After 2 hours, each flask contents were transferred to a

previously labeled 50 mL centrifuge tube and centrifuged at

2000 rpm for 15 minutes to wash the suspended cells.

2.2.4. Final Culture of CIK Cells

For the generation of CIK cells, suspended MNCs were

cultured at 37˚C and 5% CO2 in RPMI-1640 medium

containing 100 µL Pen/Strep/ Fungizone (10 µL/ 1 mL

media) and IFNγ (1,000 U/mL) on day zero. 50 ng/mL

human monoclonal anti-CD3 antibody and 300 U/mL IL-2

were added on day 1. Every 3 days, cells were counted,

examined microscopically, and maintained at a density of

1×106 cells/mL with pre-warmed complete nutrient medium

supplemented with IL-2. Fetal calf serum was not used in this

culture protocol as it has many drawbacks and leads to

serious misinterpretations in immunological studies [24, 25].

2.2.5. Suspension Culture Passage

When cultures were confluent, i.e. cells aggregate together

and the medium appears unclear when the flask is swirled

gently (~2.5 × 106 cells/mL), the culture was passaged.

Aseptically half of the volume of cell suspension was

removed and was placed into a new flask. Each flask was fed

with 7 to 10 mL pre-warmed freshly prepared complete

nutrient RPMI-1640 supplemented with IL-2.

2.3. Identification of CIK Cells

2.3.1. Morphologically

Microscopic examination of morphological changes,

growth rate, count, and viability of MNCs during culture was

carried out using an inverted microscope (Carl Zeiss®

,

Germany).

2.3.2. Phenotypically

CIK cells related surface phenotypes, CD3 and CD56,

were identified by means of flow cytometric analysis on day

0, 7, and 14 of culture by FACS Calibur (Becton Dickinson,

USA)

2.3.3. Functionally

The function of CIK cells was examined by investigation

of TNFα secretion in vitro in the culture supernatant by

ELISA assay and the cytotoxic effect of CIK cells in vitro on

HCC cell line by using cell counting kit-8 (CCK-8) assay.

CCK-8 allows a convenient assay using a water soluble

tetrazolium salt, which produces a water soluble orange

formazan dye upon bio-reduction by cellular

dehydrogenases. The amount of formazan produced is

directly proportional to the number of living cells.

2.4. Statistical Analysis

Analysis of data was performed with Statistical Package

for Social Science (SPSS) version 19. The results of

dependent variables were expressed as mean ± SD. The

overall statistical significance of the difference between the

groups’ means was assessed by one-way analysis of variance

(ANOVA) test followed by post hoc test to detect which

means pairs are statistically significantly different.

Regression between survival rate and CIK:HepG2 ratio was

done using linear regression analysis. Correlation between

measured variables was evaluated using Pearson’s correlation

coefficient. Values of P < 0.05 were considered statistically

significant.

3. Results

3.1. Morphological Characterization of CIKs

During the entire culture period, the CIK cells’ growth and

maturation were observed under the inverted microscope. On

day 4 of culture, microscopic examination of cultured MNCs

showed that the cells began to grow double in number. Induced

MNCs appeared as discrete single cells, round, and refractive to

light. The onset of cluster-like formation of CIK cells could be

observed on day 7 after first seeding of suspended MNCs. On

day 10 of culture, most of the incubated cells began to grow and

cluster together. On day 14, induced MNCs increased in number

and form suspended large clusters which were a distinctive

feature of mature CIK cells (Figure 1).

Cell Count and Viability

It was found that the separated PBMCs count ranged from

30×106 to 34×10

6cell/mL and the lymphocytes account for 60.8

to 67.6% of total separated PBMCs. The viability range upon

separation was 95–98%. ANOVA test results (Table 1, Figure 2)

showed that the effect of culture duration on MNCs density i.e.

growth rate, count, and viability was significant (P <0.001). The

growth rate of induced MNCs (Figure 2a) was significantly

increased on day 4 with culture time till reached maximum on

day 7 then there was a significant decrease over days 10 and 14

(P <0.001), while the count of induced MNCs (Figure 2b) did

not differ significantly between day zero and day 4 (P=0.291),

but there was a very statistical significant increase in count on

days 7, 10, and 14 (P <0.001). MNCs viability (Figure 2c) did

not differ significantly on day 4 (P=0.275), 7 (P=0.323), and 10

(P=0.733), but it was significantly decreased from day 10 to day

14 (P=0.031).


Figure 1. Inverted microscopic images of PBMCs. (a) Before the preliminary static culture, showing round mononuclear cells along with round biconcave red

blood cells (RBCs) contamination and irregularly shaped myeloid cells. (b) After the preliminary static culture, showing single discrete round cells with a

clear center and refractive to light (c) After 7 days, the onset of cluster-like formation of CIK cells could be observed as the suspended cells began to clump

together. (d) After 14 days, large suspended clusters of fully mature CIK cells were observed. (PBMCs images were obtained by an inverted microscope x100

(a and b) and x40 (c and d).

Table 1. Relation between density, number, and viability of induced MNCs and duration of culture. Data are presented as mean ± standard deviation. One-way

ANOVA test was applied, followed by Tukey HSD post hoc test, (P <0.05), total sample size=20. †: Overall significance effect of culture duration on the

dependent variable a: Significant increase versus the preceding time point. b: Significant decrease versus the preceding time point.

Culture duration MNCs density† (x 106 cells/mL) MNCs number† (cells/flask x106) Viability† (%)

Day 0 1 10 96.901 ± 1.50

Day 4 2.090 ± 0.100 a 20.900 ± 1 94.414 ± 0.80

Day 7 3.740 ± 0.124 a 74.800 ± 2.487 a 92.252 ± 0.93

Day 10 3.090 ± 0.139 b 123.600 ± 5.713 a 90.901 ± 1.84

Day 14 2.795 ± 0.190 b 223.600 ± 15.588 a 87.251 ± 2.38 b

Figure 2. Relation between density, number, and viability of induced MNCs and duration of culture. (a) Cell density, (b) Number of induced MNCs, (c)

Viability. Data are presented as mean ± standard deviation. One-way ANOVA test was applied, followed by Tukey HSD post hoc test, (P <0.05), total sample

size=20. a: Significant increase versus the preceding time point, b: Significant decrease versus the preceding time point.



3.2. Phenotypical Characterization of CIKs

On day zero, MNCs were positive for CD3 (6.253 ±

3.357%) and CD56 (1.737 ± 0.274%). The percentages of T

cells and NK cells were 5.867 ± 3.435 and 1.350 ± 0.226,

respectively; while the percentage of NKT cells was 0.387 ±

0.091. The results showed that the expression of both CD3

and CD56 phenotypes were significantly affected by culture

duration as determined by one-way ANOVA test (P <0.001).

The expression of CD3 was significantly increased on day 7

(21.047 ± 2.912, P=0.042) and on day 14 (72.220 ± 8.698, P

<0.001). The expression of CD56 showed more significant

increase than CD3 on day 7 compared to day zero (12.123 ±

1.625, P=0.003) and on day 14 compared to day 7 (31.203 ±

3.500, P <0.001) (Figure 3).

According to previous phenotypic results, percentages of T

cells, NK cells, and NKT cells were estimated on day 7 and

14. One-way ANOVA analysis showed an overall statistically

significant effect of culture duration on the expression of T

cells, NKs, and NKT cells (P <0.001). Tukey HSD post hoc

test (Table 2 and Figure 3) indicated that the percentage of T

cells was insignificantly increased on day 7 (P=0.084), but it

was significantly increased on day 14 (P <0.001). The

percentage of NKs was significantly increased on day 7 (P

<0.001), but it did not show a significant increase from day 7

to day 14 (P=0.523). The percentage of the most cytotoxic

subset under investigation, CD3+CD56

+, indicated that there

was a significant increase in expression of NKT cells on day

7 (6.593 ± 0.843, P=0.029) and on day 14 (25.137 ± 3.656, P

<0.001).

Figure 3. Relation between the percent of phenotype expression on induced MNCs and culture duration. Data are presented as mean ± standard deviation.

One-way ANOVA test was applied, followed by Tukey HSD post hoc test, (P <0.05), total sample size=9. a: Significant increase versus the preceding time

point.

Table 2. The percentages of T cells, NK cells, and NKT cells on days 0, 7, and 14 of culture. Data are presented as mean ± standard deviation. One-way

ANOVA test was applied, followed by Tukey HSD post hoc test, (P <0.05), total sample size=9. #: Overall significance effect of culture duration on the

dependent variable a: Significant increase versus the preceding time point.

Culture duration % T cells † % NK cells† % NKT cells†

Day 0 5.867 ± 3.435 1.350 ± 0.226 0.387 ± 0.091

Day 7 14.453 ± 2.242 5.530 ± 0.934 a 6.593 ± 0.843 a

Day 14 47.083 ± 5.516 a 6.067 ± 0.247 25.137± 3.656 a

3.3. Functional Characterization of CIK Cells

3.3.1. Tumor Necrosis Factor Alpha Secretion

Tumor necrosis factor alpha was measured in culture

supernatant at the beginning of the trial, day zero, and at the

end of it, day 14. The result of TNFα concentration, on day

zero, was Nil, i.e. undetectable. On day 14, the ELISA assay

was performed on four different samples using a microplate

reader at 450nm (Unicam®, United Kingdom). Pooled mean

± pooled standard deviation of TNFα concentration,

calculated from 4 samples in octuplicate by the following

equations, was 14.538 ± 6.672 pg/mL.

Pooled mean = ��

�� (1)

Pooled standard deviation =

�� (2)


Where N: number of test sample wells, M: mean of test

sample wells, S: standard deviation from sample mean

3.3.2. Cytotoxicity Assay of CIK Cells

The cytotoxic effect of CIKs was measured on day 14 on

mature CIK cells by using CCK-8 assay. It was investigated

on hepatocellular carcinoma HepG2 cell line in vitro. HepG2

adherent cells were trypsinized for 10 minutes and were

examined for detachment every 3–4 minutes by the inverted

microscope. When the detached HepG2 hepatocytes were

viewed as floating, compact, round with dense cytoplasm

cells, and surrounded by spindle-shaped cells, 9 mL of

RPMI-1640 + 1mL fetal bovine serum were added to

neutralize the excess trypsin.

The survival rate is a part of survival analysis that represents

the percentage of cells in a study or treatment group still

alive for a given period of time after using a certain toxic

agent. It was calculated by the following equation:

Percent survival rate of HepG2 cells =

�� X 100 (3)

Where Asample: sample wells, ACIK: CIK wells,

AHepG2: HepG2 wells, Ablank: blank wells

The hypothesis test for the null hypothesis of zero slope

between the two variables, X (CIK:HepG2 ratio) independent

variable and Y (survival rate) dependent variable. One-way

ANOVA analysis showed an overall statistically significant

effect of CIK:HepG2 ratio on the percentage of HepG2

survival rate (P <0.001). Post hoc analysis using Tukey HSD

test indicated that HepG2 survival rate at (5:1) ratio was

insignificantly decreased from (1:1) ratio, yet it was

significantly decreased at (10:1), (20:1), and (40:1) ratios (P

<0.001). The minimum survival rate of HepG2 cells was at

the CIK:HepG2 ratio of 40:1, which in turn means that the

cytotoxic effect of CIK cells at (40:1) ratio was 58.889 ±

1.104 %.

3.4. Results of Regression Analysis

Regression analysis indicated that the survival rate is

negatively correlated with the CIK:HepG2 ratio (r = –0.989)

and that regression model could statistically significantly

predict the outcome variable, the survival rate of HepG2 cells

based on CIK:HepG2 ratio (P=0.001). A Significant

regression equation was used to describe the statistical

relationship between dependent variable (Y), survival rate,

and independent variable (X), CIK:HepG2 ratio (Figure 4),

with a significant slope (P=0.001) and significant Y-intercept

(P <0.001). Since the slope of the regression line was

significantly different from zero, the null hypothesis of zero

slope between the variables could be rejected and accept the

alternative hypothesis that there is a significant relationship

between the independent and dependent variables.

The regression equation was estimated [Y = – 1.559 X +

101.027]; where (–1.559) was the slope and (101.027) was

the Y-intercept. From this equation, the CIK:HepG2 ratio

required to achieve zero survival rate of HepG2 cells could

be predicted. If Y is replaced with zero, then the X value

would be 64.8. This means that at a CIK:HepG2 ratio of

65:1, the survival rate of HepG2 cells would be zero.

Figure 4. Linear regression chart representing survival rate (%) of HepG2

cells against different ratios of CIK:HepG2 cells. One-way ANOVA test was

applied, followed by Tukey HSD post hoc test, (P <0.05), total sample

size=15. Linear regression analysis, (P <0.05), n=5. b: Significant decrease

versus the increase in CIK:HepG2 ratio.

3.5. Results of Correlation Analysis

Pearson’s correlation analysis was applied to indicate the

correlation between measured variables in the study (Table

3). Duration of CIK cells culture showed a significant

positive correlation with the number of induced MNCs

(P=0.009), T cells expression (P=0.009), NK cells expression

(P=0.025), and NKT cells expression (P=0.008) and a

significant negative correlation with the viability of induced

MNCs (P <0.001). Number of induced MNCs over the entire

culture period showed a significant positive correlation with

T cells expression (P=0.001), and NKT cells expression

(P=0.001); while there was a significant negative correlation

with the viability of induced MNCs (P=0.005). The growth

rate of induced MNCs showed a significant positive

correlation with NK cells expression (P=0.032). The viability

of MNCs showed a negative correlation with T cells

expression (P=0.009), NK cells expression (P=0.025), and

NKT cells expression (P=0.008). T cells expression showed a

positive significant correlation with the expression of NKT

cells (P <0.001).

Table 3. Results of Correlation Analysis. r: Pearson correlation coefficient, n=5. p: P value, p (2-tail) <0.05 was considered to be statistically significant. *:

Significant correlation, +: Positive correlation, −: Negative correlation.

Culture duration MNCs number MNCs density MNCs viability T cells NK cells NKT cells

Culture

duration

r +1 +0.961* 0.690 −0.995* +0.961* +0.924* +0.965*

P 0.009 0.197 < 0.001 0.009 0.025 0.008

MNCs number r +0.961* +1 0.517 −0.973* +0.989* 0.802 +0.989*



Culture duration MNCs number MNCs density MNCs viability T cells NK cells NKT cells

P 0.009 0.372 0.005 0.001 0.103 0.001

MNCs density r 0.690 0.517 +1 −0.666 0.474 +0.910* 0.489

P 0.197 0.372 0.220 0.420 0.032 0.403

MNCs viability r −0.995* −0.973* −0.666 +1 −0.963* −0.903* −0.967*

P < 0.001 0.005 0.220 0.009 0.036 0.007

T cells r +0.961* +0.989* 0.474 −0.963* +1 0.788 +0.999*

P 0.009 0.001 0.420 0.009 0.113 < 0.001

NK cells r +0.924* 0.802 +0.910* −0.903* 0.788 +1 0.799

P 0.025 0.103 0.032 0.036 0.113 0.105

NKT cells r +0.965* +0.989* 0.489 -0.967* +0.999* 0.799 +1

P 0.008 0.001 0.403 0.007 < 0.001 0.105

4. Discussion

Hepatocellular carcinoma is a disruptive cancer that occurs

as an end result of chronic liver disease and cirrhosis [4].

Malignant liver cells often survive traditional treatment such

as radiation and chemotherapy. Most importantly, small

lesions and metastatic cells often remain and cause

recurrence of disease [26]. Concomitant liver dysfunction

with advanced tumor stages further impedes curative

therapies. Thus, available treatments of this disease are

highly complex, as they not only include the tumor biology

and anatomic considerations within the liver but also the

underlying function of the liver and the patient’s functional

status [10]. Immunotherapy is a new and promising treatment

for a number of cancers. Cell-based immunotherapy is a set

of therapeutic strategies based on manipulating and co-opting

a patient’s own immune cells, or donor cells and using

immune cell functions to halt and reverse disease [27].

The present study evaluates the potential of in vitro

expansion of viable CIK cells from human PBMCs and

measures the proportion of the most effective subset

CD3+CD56

+ in the culture. In addition, the study examines

TNFα secretion and the cytotoxicity of expanded CIK cells

on HepG2 cell line. Different culturing protocols for CIK

cells were reported; yet all these protocols share the main

three growth factors pillars used in CIK culture, which are,

IFNγ, IL-2, and monoclonal anti-CD3 antibody. The

differences between them relied on different MNCs origin,

nutrient growth medium, protein serum, growth factors’

concentrations or different cytokines combinations.

In this study, the suspended subset of PBMCs, obtained

from healthy volunteers, were cultured in RPMI-1640

medium without addition of protein serum to cultured cells,

either in the form of fetal calf serum or as heat-inactivated

plasma, as the foreign proteins may lead to sensitization of

killer cells to react with them. This finding was in line with

Kerbel and Blakeslee [24] and Reddy et al. [25], who

reported that fetal calf serum should not be added to cell

cultures as it causes many serious misinterpretations in

immunological researches. Suspended cells were cultured for

14 days with IFNγ, monoclonal anti-CD3 antibody, and IL-2;

which is T cell growth factor that promotes naїve T cells to

differentiate into effector T cells, modulates differentiation of

Th cells, promotes Treg cells development, augments

cytolytic activity of NK, and regulates effector versus

memory T cell generation [28].

Allogeneic protocols were reported by Iudicone et al. [29]

and Guo et al. [30]. Autologous protocols were also reported

by Niam et al. [31], Liu et al. [32], and Chan and Linn [33],

who have obtained CIK cells from patients with myeloid

leukemia, hepatocellular carcinoma, and polycythemia,

respectively. According to Zhang et al. [34], the antitumor

efficacy of autologous CIK cells derived from cancer patients

was lower because of the immunosuppressive state of

patients, compared with the allogeneic CIK cells obtained

from adult healthy individuals. Protocols involved cord blood

MNCs differentiation into CIK cells were also reported by

Zhang et al. [35] and Durrieu et al. [36], who reported that

CIK cells differentiated from cord blood differ in some

receptors’ expression and cytotoxic pathways, suggesting that

the source of CIK cells may impact on therapeutic and

cytotoxic efficacy.

The hypothesis of the protein serum effect on cultured CIK

cells was followed by Guo et al. [30], Meng et al. [21], and

Li et al. [37]. On the contrary, Niam et al. [31] used a

complete nutrient medium of RPMI-1640 and 10% fetal calf

serum, claiming that this was the optimal medium for CIK

cell expansion, better than other serum-free MNCs culture

media, despite that, the CD3+CD56

+ effective subset reported

comprised a median of 26.6%. Wei et al. [38] and Liu et al.

[32] also used serum proteins, 10% fetal calf serum and 1%

heat-inactivated plasma, respectively, during their CIK

culturing protocols.

Other culturing protocols for CIK cells to enhance

proliferation or selective tumor toxicity with different

cytokines combinations were reported. Helms et al. [39] used

IL-12 to shorten the in vitro expansion process for CIK cells

and enhance their cytotoxic efficacy. Lin et al. [40] used IL-6

to decrease the proportion of Treg cells which inhibit cellular

immunity against the tumor. Tao et al. [41] used IL-15

instead of IL-2 for in vitro CIK cells culture in order to

enhance CIK cells–mediated cytotoxicity against leukemia.

Rajbhandary et al. [42] used IL-21 to enhance the cytotoxic

effect of CIK cells through increased expression of IFNγ,

TNFα, granzyme B, and perforin. Ingersoll et al. [43] used

IL-7 to expand cytotoxic CIK cells that improved survival in

a xenograft mouse model of ovarian cancer. Iudicone et al.

[29] used IL-15 on the 7th

day of culture instead of IL-2 to

enhance CIK cells–mediated cytotoxicity against epithelial

cancer cell lines.

During the entire culture period in the current study, the


inverted microscope was used to observe the growth and

maturation of CIK cells. The onset of proliferation may be

due to the direct effect of IL-2 that promoted naїve T cells to

differentiate into effector cells [28] and the indirect effect of

IFNγ mediated by monocytes activation, providing soluble

proliferating factors (IL-12) and the initial mitogenic signal

provided by the monoclonal anti-CD3 antibody on day 1 and

sustained by the continuous presence of IL-2 every 3 days

along the entire culture period [44]. The onset of cells

maturation, cluster-like formation, could be observed on day

7, while fully matured suspended CIK clusters could be

observed on day 14.

This goes with the results reported by Li et al. [45], who

cultured MNCs for 14 days and found that the cells began to

proliferate within 3 days and became fully matured as

colonies in suspension within 14 days. On the other hand,

these findings were not in line with Bonanno et al. [46],

Niam et al. [31], and Wei et al. [38], who cultured human

PBMCs for 21, 28, and 15 days, respectively, to reach the

maturation stage. In addition, Chan and Linn [33] cultured

human PBMCs for 26 days and they started counting and

evaluating morphological and phenotypical changes of CIK

cells on day 10 as they assumed that it is too early to assess

cultured CIK cells before day 10.

In the present study, the count and viability of MNCs were

reported every 3 days, before adjusting cell density. The

results showed a significant increase in cells number with

culture duration. This finding was not in line with Niam et al.

[31], who reported that the number of CIK cells dropped to a

median of 0.44 fold of starting number at the first counting at

day 11 of culture, after which growth started from about day

14 and approached a plateau by day 28. The reported drop in

number in his study was likely to be as a result of

supplementing cell culture with more complete media and

IL-2 at day 7, without adjustment to cell density. Thus, the

crowded cell media and increased waste products led to cells

death. When the cell density had been adjusted, from day 10

onward, the cells grew and showed obvious growth count

from about day 14. The viability of induced PBMCs in this

study was decreased on day 14 significantly with time to

87.251 ± 2.38%. These results go in compliance with Luo et

al. [47], who cultured CIK cells from isolated PBMCs with

cell viability (> 90%) and reported cell viability, about 2

weeks later, to be (> 85%).

This study aims at measuring the proportion of the most

effective subset CD3+CD56

+ in the culture. The results of

CD3 and CD56 phenotypes showed a very significant

increase in expression versus culture duration. This finding

was supported by Mata-Molanes et al. [17], who reported

that after 14 days of culture, the percentage of CD3+CD56

+

subset reaches 20 to 30% of the total CIK cells. Guo et al.

[30], who cultured CIK cells from healthy volunteers’ blood

donors for 14 days, reported CD3+CD56

+ proportion on day

14 as 25.31 ± 7.42%. Li et al. [37], who cultured CIK cells

from patients with early-stage melanoma blood for 14 days,

reported CD3+CD56

+ proportion on day 14 as 21.8 ± 8%. On

the other hand, these results did not go in line with Bonanno

et al. [46], who cultured PBMCs with different

concentrations of anti-CD3 antibody (50, 250, and 250

ng/mL) reported that the proportion of CD3+CD56

+ subsets

on day 21 were 69.6%, 47.9%, and 29.3%, respectively.

The functional assays for CIK cells include the production

of cytokines and the CIK cells’ cytotoxic effect on HepG2

cell line in vitro. TNFα is one of the main cytokines produced

by CIK cells and should be fit as a test for cytokines secreted

by CIK cells [21]. TNFα can transmit apoptotic signals inside

cells that can be killed by ligand binding. It is believed that

TNFα usually gives the signal for cell survival; however, due

to other signals, some tumor cells can undergo apoptosis

[48].

In the present work, TNFα concentration, on day 14, was

14.538 ± 6.672 pg/mL. The present result was supported by

Zhang et al. [34], who investigated the secretion of TNFα

from expanded umbilical cord – CIK cells. The reported

result was much less than that of the present study (6 ± 5.5

pg/mL); which may be due to different CIK cells origin [35]

or the usage of fetal calf serum that affect immunological

studies [24, 25].

The CIK cells’ cytotoxic effect on cancerous cells showed

variable degrees of efficacy in several tumors including

malignant lymphoma either Hodgkin’s disease or non-

Hodgkin’s lymphoma [49], hematological malignancies as

acute myeloid and acute lymphocytic leukemia [50] and

chronic lymphocytic leukemia [51]. Recent in vitro studies

had further shown the potential activity of CIK cells

differentiated from the blood of healthy or patient volunteers

against breast cancer [30], pancreatic cancer [52], sarcomas

[53], ovarian cancer [44], metastatic melanoma [54],

glioblastoma or brain cancer [55], solid tumors [45], and

gallbladder cancer [56]. In this study, the cytotoxic effect of

CIK cells was investigated on HCC in vitro. The results

showed a significant cytotoxic effect of CIK cells on HepG2

cells. We also could predict the CIK:HepG2 ratio required to

achieve a complete cytotoxicity HepG2 cells, that is, 65:1.

5. Conclusion

Collectively, the present study has provided data to support

the ongoing practice of generating CIK cells from human PB,

which is an important and promising strategy for future work

involving HCC immunotherapy. PB-derived MNCs can

differentiate into CIK cells in vitro when cultured in

complete nutrient media containing IFNγ, anti-CD3 antibody,

and IL-2. The CIK culture showed different proportions of

effector and cytotoxic subsets T cells, NK cells, and NKT

cells. CIK cells showed high functional capacity as

evidenced by secretion of cytokine TNFα and cytotoxicity

against HCC cell line, HepG2. This study provides a simple

and easily handled strategy for in vitro differentiation of

human PB-derived MNCs into CIK cells. Further studies are

also needed to address the in vivo anti-cancer immune

response, toxicity of CIK cells to normal and cancerous cells,

and the predictive value of many other factors related to

malignant cells or tumor microenvironment.



Disclosure of Interest

The authors declare no conflict of interest. This research

did not receive any specific grant from funding agencies in

the public, commercial, or not-for-profit sectors.

References

[1] Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer. 2015; 136 (5): E359-86. doi: 10.1002/ijc.29210.

[2] van Ginneken V, de Vries E, Verheij E, et al. Potential Biomarkers for ‘Fatty Liver’(Hepatic Steatosis) and Hepatocellular Carcinoma (HCC) and an explanation of their pathogenesis. Gastroenterology and Liver Clinical and Medicals. 2017; 1 (1): 1-14.

[3] Dhanasekaran R, Bandoh S, Roberts LR. Molecular pathogenesis of hepatocellular carcinoma and impact of therapeutic advances. F1000Research. 2016; 5 (879). doi: 10.12688/f1000research.6946.1.

[4] Ghouri YA, Mian I, Rowe JH. Review of hepatocellular carcinoma: Epidemiology, etiology, and carcinogenesis. Journal of carcinogenesis. 2017; 16 (1). doi: 10.4103/jcar.JCar_9_16.

[5] Agosti P, Sabbà C, Mazzocca A. Emerging metabolic risk factors in hepatocellular carcinoma and their influence on the liver microenvironment. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2018;1864(2):607-617. doi: 10.1016/j.bbadis.2017.11.026.

[6] Horst AK, Neumann K, Diehl L, et al. Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells. Cellular & molecular immunology. 2016;13 (3): 277-292. doi: 10.1038/cmi.2015.112.

[7] Ma W, Chen X, Yuan Y. T-cell-associated immunotherapy: a promising strategy for the treatment of hepatocellular carcinoma. Immunotherapy. 2017; 9 (7): 523-525. doi: 10.2217/imt-2017-0053.

[8] Sachdeva M, Chawla YK, Arora SK. Immunology of hepatocellular carcinoma. World Journal of Hepatology. 2015; 7 (17): 2080-2090. doi: 10.4254/wjh.v7.i17.2080.

[9] Parker KH, Beury DW, Ostrand-Rosenberg S. Myeloid-Derived Suppressor Cells: Critical Cells Driving Immune Suppression in the Tumor Microenvironment. Advances in cancer research. 2015; 128: 95-139. doi: 10.1016/bs.acr.2015.04.002.

[10] Orcutt S, A. Anaya D. Liver Resection and Surgical Strategies for Management of Primary Liver Cancer. Cancer Control. 2018; 25 (1). doi: 10.1177/1073274817744621

[11] Rich NE, Yopp AC, Singal AG. Medical Management of Hepatocellular Carcinoma. Journal of oncology practice. 2017; 13 (6): 356-364. doi: 10.1200/jop.2017.022996.

[12] Baradaran Noveiry B, Hirbod-Mobarakeh A, Khalili N, et al. Specific immunotherapy in hepatocellular cancer: A systematic review. Journal of gastroenterology and hepatology. 2017; 32 (2): 339-351. doi: 10.1111/jgh.13449.

[13] Vu BT, Duong QT-N, Le PM, et al., editors. Culture and differentiation of cytokine-induced killer cells from umbilical cord blood-derived mononuclear cells. International Conference on the Development of Biomedical Engineering in Vietnam; 2017: Springer.

[14] Lim O, Jung MY, Hwang YK, et al. Present and Future of Allogeneic Natural Killer Cell Therapy. Frontiers in Immunology. 2015; 6: 286. doi: 10.3389/fimmu.2015.00286.

[15] Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 2015; 348 (6230): 62-68. doi: 10.1126/science.aaa4967.

[16] Introna M, Correnti F. Innovative Clinical Perspectives for CIK Cells in Cancer Patients. International Journal of Molecular Sciences. 2018; 19 (2): 358-371. doi: 10.3390/ijms19020358.

[17] Mata-Molanes JJ, Sureda González M, Valenzuela Jiménez B, et al. Cancer Immunotherapy with Cytokine-Induced Killer Cells. Targeted Oncology. 2017; 12 (3): 289-299. doi: 10.1007/s11523-017-0489-2.

[18] Introna M. CIK as therapeutic agents against tumors. Journal of Autoimmunity. 2017; 85: 32-44. doi: 10.1016/j.jaut.2017.06.008.

[19] Cappuzzello E, Sommaggio R, Zanovello P, et al. Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells. Cytokine & Growth Factor Reviews. 2017; 36: 99-105. doi: 10.1016/j.cytogfr.2017.06.003.

[20] Guo Y, Han W. Cytokine-induced killer (CIK) cells: from basic research to clinical translation. Chinese Journal of Cancer. 2015; 34 (3). doi: 10.1186/s40880-015-0002-1.

[21] Meng Y, Yu Z, Wu Y, et al. Cell-based immunotherapy with cytokine-induced killer (CIK) cells: From preparation and testing to clinical application. Human Vaccines & Immunotherapeutics. 2017; 13 (6): 1379-1387. doi: 10.1080/21645515.2017.1285987.

[22] Janetzki S. Sample Preparation. In: Janetzki S, editor. Elispot for Rookies (and Experts Too). Cham: Springer International Publishing; 2016. p. 25-41.

[23] Riedhammer C, Halbritter D, Weissert R. Peripheral Blood Mononuclear Cells: Isolation, Freezing, Thawing, and Culture. Methods in molecular biology. 2016; 1304:53-61. doi: 10.1007/7651_2014_99.

[24] Kerbel RS, Blakeslee D. Rapid adsorption of a foetal calf serum component by mammalian cells in culture. A potential source of artifacts in studies of antisera to cell-specific antigens. Immunology. 1976; 31 (6): 881-891.

[25] Reddy BP, Reddy BP, Rayulu DJ. Effects of fetal bovine serum concentration on the growth and survival of BHK 21 cell lines. International Journal of Applied Biology and Pharmaceutical Technology. 2016; 7 (2): 122-127.

[26] Hontscha C, Borck Y, Zhou H, et al. Clinical trials on CIK cells: first report of the international registry on CIK cells (IRCC). Journal of Cancer Research and Clinical Oncology. 2011; 137 (2): 305-310. doi: 10.1007/s00432-010-0887-7.

[27] Schmidt TL, Negrin RS, Contag CH. A killer choice for cancer immunotherapy. Immunologic Research. 2014; 58 (2): 300-306. doi: 10.1007/s12026-014-8507-2.


[28] Mitra S, Ring AM, Amarnath S, et al. Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps. Immunity. 2015; 42 (5): 826-838. doi: 10.1016/j.immuni.2015.04.018.

[29] Iudicone P, Fioravanti D, Cicchetti E, et al. Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway. Human Immunology. 2016; 77 (12): 1239-1247. doi: 10.1016/j.humimm.2016.09.003.

[30] Guo Q, Zhu D, Bu X, et al. Efficient killing of radioresistant breast cancer cells by cytokine-induced killer cells. Tumour Biology. 2017; 39 (3). doi: 10.1177/1010428317695961.

[31] Niam M, Linn YC, Fook Chong S, et al. Clinical scale expansion of cytokine-induced killer cells is feasible from healthy donors and patients with acute and chronic myeloid leukemia at various stages of therapy. Experimental hematology. 2011; 39 (9): 897-903. doi: 10.1016/j.exphem.2011.06.005.

[32] Liu H, Li J, Wang F, et al. Comparative study of different procedures for the separation of peripheral blood mononuclear cells in cytokine-induced killer cell immunotherapy for hepatocarcinoma. Tumour biology. 2015; 36 (4): 2299-3307. doi: 10.1007/s13277-014-2837-5.

[33] Chan W-C, Linn Y-C. A comparison between cytokine-and bead-stimulated polyclonal T cells: the superiority of each and their possible complementary role. Cytotechnology. 2016; 68 (4): 735-748. doi: 10.1007/s10616-014-9825-x.

[34] Zhang Z, Zhao X, Zhang T, et al. Phenotypic characterization and anti-tumor effects of cytokine-induced killer cells derived from cord blood. Cytotherapy. 2015;17 (1): 86-97. doi: 10.1016/j.jcyt.2014.09.006.

[35] Zhang Q, Wang L, Luo C, et al. Phenotypic and functional characterization of cytokine-induced killer cells derived from preterm and term infant cord blood. Oncology Reports. 2014; 32 (5): 2244-2252. doi: 10.3892/or.2014.

[36] Durrieu L, Lemieux W, Dieng MM, et al. Implication of different effector mechanisms by cord blood-derived and peripheral blood-derived cytokine-induced killer cells to kill precursor B acute lymphoblastic leukemia cell lines. Cytotherapy. 2014; 16 (6): 845-56. doi: 10.1016/j.jcyt.2013.12.010.

[37] Li H, Huang L, Liu L, et al. Selective effect of cytokine-induced killer cells on survival of patients with early-stage melanoma. Cancer immunology, immunotherapy. 2017;66(3):299-308. doi: 10.1007/s00262-016-1939-x.

[38] Wei C, Wang W, Pang W, et al. The CIK cells stimulated with combination of IL-2 and IL-15 provide an improved cytotoxic capacity against human lung adenocarcinoma. Tumour biology. 2014; 35 (3): 1997-2007. doi: 10.1007/s13277-013-1265-2.

[39] Helms MW, Prescher JA, Cao Y-A, et al. IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy. Cancer immunology, immunotherapy. 2010; 59 (9): 1325-1334. doi: 10.1007/s00262-010-0860-y.

[40] Lin G, Wang J, Lao X, et al. Interleukin-6 inhibits regulatory T cells and improves the proliferation and cytotoxic activity of cytokine-induced killer cells. Journal of immunotherapy. 2012; 35 (4): 337-343. doi: 10.1097/CJI.0b013e318255ada3.

[41] Tao Q, Chen T, Tao L, et al. IL-15 improves the cytotoxicity of cytokine-induced killer cells against leukemia cells by upregulating CD3+CD56+ cells and downregulating regulatory T cells as well as IL-35. Journal of immunotherapy. 2013; 36 (9): 462-467. doi: 10.1097/cji.0000000000000001.

[42] Rajbhandary S, Zhao M-F, Zhao N, et al. Multiple cytotoxic factors involved in IL-21 enhanced antitumor function of CIK cells signaled through STAT-3 and STAT5b pathways. Asian Pacific Journal of Cancer Prevention. 2013; 14 (10): 5825-5831.

[43] Ingersoll SB, Srivastava M, Ali G, et al. Cytokine-induced killer cells from ovarian cancer patients expanded ex vivo in the presence of IL-7 improve survival in a xenograft mouse model of ovarian cancer. Gynecologic Oncology. 2014; 133: 120-121. doi: 10.1016/j.ygyno.2014.03.316.

[44] Mesiano G, Todorovic M, Gammaitoni L, et al. Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors. Expert opinion on biological therapy. 2012; 12 (6): 673-84. doi: 10.1517/14712598.2012.675323.

[45] Li GX, Zhao SS, Zhang XG, et al. Comparison of the proliferation, cytotoxic activity and cytokine secretion function of cascade primed immune cells and cytokine-induced killer cells in vitro. Molecular Medicine Reports. 2015; 12 (2): 2629-2635. doi: 10.3892/mmr.2015.3765.

[46] Bonanno G, Iudicone P, Mariotti A, et al. Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures. Journal of Translational Medicine. 2010; 8 (1): 129. doi: 10.1186/1479-5876-8-129.

[47] Luo H, Gong L, Zhu B, et al. Therapeutic outcomes of autologous CIK cells as a maintenance therapy in the treatment of lung cancer patients: A retrospective study. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2016; 84: 987-993. doi: 10.1016/j.biopha.2016.10.022.

[48] Dembic Z. Chapter 8 - Cytokines Important for Growth and/or Development of Cells of the Immune System. In: Dembic Z, editor. The Cytokines of the Immune System. Amsterdam: Academic Press; 2015. p. 263-281.

[49] Guo ZHI, Liu HAO, He X-P, et al. A clinical study of cytokine-induced killer cells for the treatment of refractory lymphoma. Oncology Letters. 2011; 2 (3): 531-536. doi: 10.3892/ol.2011.269.

[50] Linn YC, Niam M, Chu S, et al. The anti-tumour activity of allogeneic cytokine-induced killer cells in patients who relapse after allogeneic transplant for haematological malignancies. Bone marrow transplantation. 2012; 47 (7): 957-966. doi: 10.1038/bmt.2011.202.

[51] Kornacker M, Moldenhauer G, Herbst M, et al. Cytokine-induced killer cells against autologous CLL: direct cytotoxic effects and induction of immune accessory molecules by interferon-gamma. International journal of cancer. 2006; 119 (6): 1377-82. doi: 10.1002/ijc.21994.

[52] Youlin K, Jian K, Siming L, et al. Potent anti-prostate cancer immune response induced by dendritic cells transduced with recombinant adenoviruses encoding 4-1BBL combined with cytokine-induced killer cells. Immunotherapy. 2015; 7 (1): 13-20. doi: 10.2217/imt.14.92.



[53] Sangiolo D, Mesiano G, Gammaitoni L, et al. Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas. Cancer Research. 2014; 74 (1): 119-129. doi: 10.1158/0008-5472.CAN-13-1559.

[54] Gammaitoni L, Giraudo L, Leuci V, et al. Effective Activity of Cytokine-Induced Killer Cells against Autologous Metastatic Melanoma Including Cells with Stemness Features. Clinical Cancer Research. 2013; 19 (16): 4347-4358. doi: 10.1158/1078-0432.CCR-13-0061 %J Clinical Cancer Research.

[55] Jin J, Joo KM, Lee SJ, et al. Synergistic therapeutic effects of cytokine-induced killer cells and temozolomide against glioblastoma. Oncology Reports. 2011; 25 (1): 33-39. doi: 10.3892/or_00001038.

[56] Wang J, He M, Shi W, et al. Inducible costimulator (ICOS) enhances the cytolytic activity of cytokine-induced killer cells against gallbladder cancer in vitro and in vivo. Cancer investigation. 2009 Mar; 27 (3): 244-250. doi: 10.1080/07357900802239124.

cytokine-induced killer cells as an adoptive cellular...

Documents